1. Field of the Invention
This invention relates to methods for chemically modifying organic compounds to form haptens chemically bonded to immunogenic substances. The invention further relates to immunological methods to determine the presence or concentration of organic compounds and, in particular, spectinomycin.
2. Description of the Background
Spectinomycin (decahydro-4a,7,9-trihydroxy-2-methyl-6,8-bis(methylamino)-4H-pyrano[2,3-b ][1,4]benzodioxin-4-one) is an aminocyclitol antibiotic isolated from the fermentation broth of Streptomyces spectabilis (D. J. Mason et al., Antibiot. Chemotherapy 11:118, 1961; E. M. Bergy et al., Antibiot. Chemotherapy 11:661, 1961). The current standard for analysis is a microbiological assay (Marsh et al., J. Assoc. Offic. Anal. Chem. 50:457, 1967; A. W. Neff et al., J. Assoc. Off. Anal. Chem. 56:834, 1973). Although cumbersome, labor intensive and slow, these methods have served as the benchmark for nearly thirty years and are the basis for the U.S. Pharmacopoeia guidelines as well as the World Health Organization analytical standards. Recently this methodology was modified to incorporate a trifluoroacetic acid extraction step (G. L. Stahl et al., J. Assoc. Off Anal. Chem. 74:471, 1995).
To improve the detectability of any analyte, the analytical paradigm suggests that the analyte can be modified to enhance its detectability, for example, chemical derivatization to introduce a chromaphore onto the analyte. Similarly, the use of a detection device that is more sensitive to the analyte, for example, fluorimetric versus colorimetric analysis will also enhance detectability. In the alternative, removal of interfering substances through the use of chromatographic separations for instance, will enhance the detectability of the analyte.
Spectinomycin represents one of the more challenging analytical problems of detectability due, in part, to the absence of a chromaphore and virtual insolubility of spectinomycin in organic solvents. As such, Spectinomycin represents a broad class of organic compounds being thermally and/or hydrolytically labile and for which improved methods of analysis are desired. To improve the detectability of spectinomycin, several approaches have been attempted over the years. Elrod employed an electrochemical detector after separation of the spectinomycin and spectinomycin-related residues by HPLC (L. Elrod et al., Pharm. Res. 5:664, 1988). Phillips used pulsed amperometric detection after cation exchange chromatography (J. G. Phillips et al., J. Chromatog. 675:123, 1994). Abbay used dynamic headspace gas chromatographic analysis of bulk spectinomycin hydrochloride (G. N. Abbay et al., LC-GC 9:100, 1991). Medina used thin layer chromatography for the semi-quantitative analysis of derivatized aminocyclitols including spectinomycin (M. B. Medina, J. Chrom. 663:127, 1995). Other derivatization strategies, followed by chromatographic analysis, have been employed by us and others. For instance, amine derivatizing agents such as 1-fluoro-2,4-nitrophenyl-5-alanine amide (FDAA or Marfey's Reagent) or 9-fluorenylmethyl chloroformate (Fmoc-Chloride) have been used to derivatize spectinomycin prior to HPLC analysis with either UV or fluorescence detection respectively. Similarly, spectinomycin has been derivatized as the trifluoromethyl ethers, trifluoroacetyl esters, and trimethylsilyl ethers, followed by gas chromatographic analysis and electron capture detection.
Immunochemical methods of analysis address the analytical paradigm in two ways. First, the antibodies themselves are highly specific for the antigen (analyte), and second, either the antigen, the antibody or an antiglobulin may be conjugated to an enzyme that produces an intensely colored or fluorescent product in the presence of the enzyme substrate to enhance the detectability. Various formats for immunochemical assays have been employed in either competitive or non-competitive assays with the detector response being either directly or indirectly proportional to the analyte concentration (J. Clausen, Immunochemical Techniques for the Identification and Estimation of Macromolecules, 3rd revised edition, R. H. Burdon and P. H. Knippenberg eds., Elsevier Science Publishers, New York, 1991).
Low molecular weight compounds such as spectinomycin are not typically antigenic and, therefore, such compounds are not capable of eliciting an immune response by themselves. The chemistry of methods utilized to conjugate small molecules to proteins and other immunogenic substances has been extensively described by Clausen as well as by Wong and the references therein (S. S. Wong, Chemistry of Protein Conjugation and Cross-Linking, CRC Press, Inc., Boca Raton, 1993). Heterocyclic compounds, including aminocyclitols like spectinomycin, require additional chemical modification prior to conjugation to a protein or other immunogenic substance. The chemistry for the addition of a four-carbon carboxyl spacer arm via condensation with succinic anhydride to form succinyl esters was first described for estrogenic steroids (Tulchinsky et al., J. Clin. Endocrinol. Metab. 33:775, 1971; Wu et al., Steroids 18:91, 1971; Emment et al., Acta Endocrinol. 69:567, 1972). These reactions required several hours of incubation or reflux conditions that would degrade a thermally labile compound such as spectinomycin or would result in multiple products.
Thus, there exists a need for a method of analysis to determine spectinomycin that is at once specific, sensitive, reliable, quantitative and inexpensive to perform. Whereas immunochemical methods of analysis could potentially fulfill these requirements, there also exists a need for a method to convert thermally and hydrolytically labile compounds such as spectinomycin into an immunogen by chemical derivatization at ambient or sub-ambient temperatures and at or near neutral pH conditions with efficient coupling to an immunogenic substance.